Abstract OT2-19-01: Single cell characterization of longitudinal biopsies from breast cancer patients treated neoadjuvantly with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib in concert
Authors: Marie Fongård, Chloé Steen, Salim Ghannoum, Marius Bjørnstad, Barbro Holm, Tatjana Bosnjak, Laurens Reitsma, Stephanie Geisler, Kamilla Fjermeros, Johannes Bruteig, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Marie Loeng, Aino Vuoriluoto, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli
Journal: Cancer Research (2023)

Abstract Background The recent introduction of CDK4/6 inhibitors has been one of the most pivotal breakthroughs in breast cancer therapy during the last decades. A growing body of evidence proposes that CDK4/6 inhibitors influence the recruitment of immune cells in the tumor microenvironment with potential effects on the outcome. Study design The NeoLetRib-study is a multicenter, single-arm, open-label, neoadjuvant, phase II trial aiming at treating 100 locally advanced luminal-A and luminal-B breast cancer patients, defined as either large T2, or T3/T4, and/or N2-3. Patients receive neoadjuvant therapy for 6 months: ribociclib (600 mg daily, 21 days on/7 days off) and letrozole (2.5 mg daily). Peri- and premenopausal women also receive therapy with goserelin 3.6 mg s.c every 28 days. Methods Three sequencial tumor biopsies were collected: pre-treatment and on-treatment (cycle 1 - day 21 and cycle 6 - day 21 with ribociclib). These biopsies were subjected to single-cell transcriptome, T cell receptor and B cell receptor profiling using the Chromium Single-Cell v2 5′ Chemistry (10x Genomics). Libraries were paired-end sequenced on a NovaSeq6000. Single cell gene expression matrices were analyzed with the Seurat package (v4.0.2). After filtering out stressed/dying cells or cells with low quality sequencing, gene expression of the remaining good quality cells was normalized and scaled to construct principal components and further cluster cells. Results In this planned interim analysis, we clustered 242315 cells from longitudinal tumor biopsies from 18 patients at pre-treatment, 18 at cycle 1 - day 21 and 9 at cycle 6 - day 21, respectively. We identified 8 main cell types: T cells, B cells, epithelial cells, fibroblasts, endothelial cells, macrophages, mast cells and dendritic cells. To further identify specific and specialized cell subtypes, we clustered the cells belonging to the above-mentioned cell types and annotated the clusters obtained using validated marker genes. Statistical methods and algorithms were then used to characterize how the proportion of different cell types changes in tumors under treatment pressure. We identified significant changes in immune cell proportions, including regulatory T cells, CD14 monocytes, SLC2A1-Macrophages among others. Conclusions In this unique patient cohort, we used single cell transcriptome profiling to obtain a high-resolution map of cell types found in tumor biopsies from the NeoLetRib trial. We characterized the effects of the combination of ribociclib and letrozole on the tumor microenvironment and identified cells sensitive and resistant to treatment. In this interim analysis, the observed longitudinal changes of immune cell types proportions in the tumor microenvironment might suggest immune related effects of the treatment combination. Citation Format: Marie Fongård, Chloé Steen, Salim Ghannoum, Marius Bjørnstad, Barbro Holm, Tatjana Bosnjak, Laurens Reitsma, Stephanie Geisler, Kamilla Fjermeros, Johannes Bruteig, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Marie Loeng, Aino Vuoriluoto, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Single cell characterization of longitudinal biopsies from breast cancer patients treated neoadjuvantly with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib in concert [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-19-01.